Comparative 16S ribosomal RNA sequencing was used to infer natural relationships among selected mycobacteria and related organism, with emphasis on members of the Mycobacterium avium-complex (MAC). All mycobacteria examined formed a closely related (average similarity $>$95%) and exclusive assemblage. Growth rate, thermotolerance, and virulence were shown to be phylogenetically restricted traits. The slow-growers formed a coherent line of descent, distinct from the rapid-growing mycobacteria. The analysis supported the species distinction for M. avium and M. intracellulare, the identification of M. paratuberculosis as a variant of M. avium, and could not distinguish the mycobacterium isolated from a Crohn's patient from the M. avium group. The 16S rRNA database served for the design of a series of group- and species-specific oligonucleotide probes and polymerase chain reaction (PCR) primers for the detection and identification of mycobacteria. To increase sensitivity of detection of mycobacteria in natural samples, five sets of rRNA-targeted PCR primers were designed to amplify various probe target sites. Like the probes, the primer sets were designed to amplify either limited or more inclusive phylogenetically coherent assemblages of mycobacteria. Various combinations of phylogenetically-based PCR primers and probes were then applied to the detection of M. paratuberculosis in bovine fecal samples. The use of PCR amplification combined with radiolabeled probes could detect as little as 1 fg of M. paratuberculosis nucleic acid in 500 ng of fecal nucleic acid and as few as 100 M. paratuberculosis cells per gram of feces. Probe diagnosis of 117 known infected (40 animals) and noninfected (77 animals) cattle resulted in 87.5% sensitivity and 75.3% specificity when probe-suspicious animals were considered positive (20% of infected and 24.7% of uninfected animals were categorized as suspicious using the probe) and was more sensitive than fecal culture, ELISA, or a DNA-targeted probe test for paratuberculosis. In a longitudinal study of an infected dairy herd, the probe test detected 20 of 26 subclinically infected animals (compared to 17 detected by ELISA and 8 detected by fecal culture) and detected subclinically infected animals significantly earlier than either ELISA or fecal culture. These results indicate the potential of probe-based diagnosis of paratuberculosis. In addition, the use of phylogenetically-based PCR primers and probes offers flexibility in the application of this detection format for the characterization of other mycobacteria in both culture and clinical samples. (Abstract shortened with permission of author.)